DESCRIPTION: Human astroviruses are increasingly recognized as a cause of acute gastroenteritis. Self-limiting viral infection occurs specifically in intestinal epithelial cells. Human astroviruses are the prototype of the Astroviridae, a new family of nonenveloped, positive-strand RNA viruses. Recent advances in the astrovirus field, including the assembly of a full-length cDNA clone from which infectious RNA can be transcribed, have made it possible to begin a more detailed study of the viral life cycle. The broad, long-term objectives of this work are: 1) to understand the pathogenic mechanisms of astroviral infection, 2) to develop human astrovirus as a vector for foreign gene delivery to the intestinal tract, and 3) to improve detection assays for human astroviruses based on this new knowledge. These objectives are complementary and will be approached by manipulating the genome length cDNA clone of human astrovirus serotype I (H-Ast1) to define molecular features of H-Ast1 that are critical to replication, expression, and packaging. The specific aims of this proposal are: 1) identification of the cis- and trans-acting signals for replication, transcription and packaging, by studying rescue of expression of a reporter gene contained within a defective H-Ast1 genome, in the presence of functional astrovirus. A cDNA copy of such a helper-dependent reporter construct will then be used to stably transform a permissive cell line, to develop a rapid, cell-based detection system for human astrovirus; 2) characterization of the subcellular localization of nascent capsid protein, by determining the kinetics of H-Ast1 capsid protein expression and its subcellular distribution using reporter gene constructs, and studying the role of the viral encoded protease and putative nuclear localization signal in processing and intracellular localization of the capsid protein; 3) definition of the optimal parameters for the use of astrovirus as a eukaryotic expression vector, including size of the vector RNA and translational strategy for the expression of the foreign gene, and development of a packaging system for the vector. The proposed studies will likely provide insight into molecular pathogenesis of astrovirus infection (aims 1 and 2) and the feasibility of developing an astrovirus-based expression vector (aim 3). The rapid detection assay for astrovirus infection (aim #1) has broad applications, including simplifying plaque assays and facilitating epidemiological surveys.